The object of this proposal is the synthesis of a third generation of compounds identified via two iterative processes of synthesis, biological evaluation and X-ray crystal structure determinations as potent and selective inhibitors of dihydrofolate reductase (DHFR) from P. carinii (pc), T. gondii (tg) (and M. avium (ma)) and as potential clinical candidates for the treatment of infections caused by these organisms in patients with AIDS. The analogues in this study were designed on the X-ray crystal structures of previous first and second generation compounds with pcDHFR. The synthesis of the analogues is proposed via modifications of procedures from our laboratory and by established literature methods. The analogues will be evaluated as inhibitors of pcDHFR, tgDHFR and maDHFR with rat liver (rl) and human DHFR as reference enzymes. Initially, 16 analogues of the 36 proposed analogues will be synthesized and evaluated. The selectivity and potency will determine if other analogues in each Series I-IV will be synthesized or not synthesized. Thus synthetic targets will be determined by the biological activity. Potent and/or selective analogues will be further evaluated against P. carinii, T. gondii, M. avium and human foreskin fibroblast cells in culture (for cytotoxicity) and subsequently in mouse infection models of P. carinii and T. gondii. The biological evaluations will be carried out on a collaborative basis. If a problem is encountered with any of the analogues with respect to solubility, a reformulation of the analogue in different salt forms will be prepared to optimize in vivo activity as a prelude to clinical trials. Preformulation and formulation studies to afford the most appropriate route of administration and dose are proposed which includes in vitro and in vivo biopharmaceutic and pharmacokinetic characterization of selected analogues. For the first year two analogues previously identified as active in in vivo studies, will be developed to further improve effectiveness of these analogues in vivo. X-ray crystal structures of selected analogues with DHFRs will also be determined collaboratively. This study will provide third generation anti-opportunistic agents with selectivity and potency against pcDHFR, tgDHFR and maDHFR and should provide selective, less toxic, clinically useful agents, which overcome the drawbacks of currently, used regimens. Screening against tuberculosis (NIAID) and tumor cells in culture (NCI) is also proposed.